Synthetic oligonucleotides and their modified analogous are finding numerous applications in diagnostics, therapeutics, molecular biology and agricultural sciences (Beaucage and Iyer, Tetrahedron, 1992, 48, 2223; Pon et al., BioTechniques, 1994, 17, 526; Agrawal, Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs. Humana Press, Totowa, N.J., Vol. 20, 1993; Eckstein, Oligonucleotides and Analogues: A Practical Approach. IRL Press, Oxford, UK, 1992). Recently, the use of modified oligonucleotides as antigene as well as antisense agents (inhibitors of gene expression at transcription and translation stages) has opened up the doors to design and develop cost effective and rapid protocols for their synthesis and purification. In order to meet the current requirement of the oligonucleotides, the present day methodology is not viable and requires considerable attention to develop rapid and cost-effective methods. Recently, with the advent of labile protecting groups (phenoxyacetyl for adenine and guanine and isobutyryl or acetyl for cytosine; dimethylformamidine for adenine, guanine and cytosine; p-tert-butylphenoxyacetyl for adenine, cytosine and guanine), better coupling reagents, shorter coupling times, faster deprotection conditions, universal polymer supports and automated systems for their assembly have simplified the synthesis of a large number of oligonucleotides. However, for their use as therapeutics in future drug arena, the current need of the hour is to develop economical methods for assembly of such a large number of oligonucleotides in large quantities. Since solid phase oligonucleotide synthesis is, now-a-days, the most preferred method for their assembly, the polymer support itself accounts for almost one third of the total cost of the production of these molecules. Et would be of a great impact if the cost of the polymer support alone could be brought down. In our opinion, this could be done in three ways, viz., (i) to employ high loading polymer supports, where large quantities of oligonucleotides could be assembled on a relatively smaller amount of the support, (ii) to synthesize multiple oligonucleotides in tandem, and (iii) to employ reusable polymer support, so that it could be recycled for carrying out multiple oligonucleotide synthesis.
Conventional Method for Oligonucleotide Synthesis and Deprotection: (FIG. 1)
FIG. 1 describes conventional method for oligonucleotide synthesis and deprotection. Initial step comprises of reacting appropriately protected 2′-deoxyribonucleoside with a derivative of succinic acid (i.e. succinic anhydride) in the presence of a hypernucleophilic catalyst such as 4-dimethylaminopyridine (DMAP) to yield an appropriately protected 2′-deoxyribonucleoside-3′-O-succinate. In the subsequent reaction, this intermediate is reacted with aminoalkylated controlled pore glass in the presence of a suitable condensing reagent to obtain polymer supported leader nucleoside. In the FIG. 1, DMTr represents 4,4′-dimethyoxytrityl, B, a nucleobase and R is a protecting group (conventional/labile) as discussed above. The resulting polymer support after capping of residual amino groups is then used for conventional oligonucleotide synthesis, in an automated synthesizer, to produce an oligonucleotide of desired sequence attached to the linker arm. After synthesis of desired sequence, the chains are cleaved from the support under alkaline conditions. This step generates fully deprotected oligonucleotide and an amide-terminated polymer support, which can't be regenerated to another cycle of synthesis and hence goes waste.
The conventional methods are based mainly on aminoalkylated controlled pore glass (CPG), where the leader nucleoside is pre-attached via its 3′-hemisuccinate moiety, and the labile linkage is cleaved from the support with aqueous ammonium hydroxide treatment. This methodology suffers from a drawback that the polymer support, after a single use, is rendered unsuitable for refunctionalization for the next cycle of oligonucleotide synthesis. This problem has partly been addressed in recent reports, including those from the inventors' laboratory (Guzaev and Manoharan, J. Am. Chem. Soc. 2003, 125, 2380: Kumar et al., Nucl. Acids Res., 2002, 30, e130; ibid, 1999, 27, e2; Azhayev, Tetrahedron. 1999, 55, 787: Ghosh et al., J. Ind. Chem. Soc., 1998, 75, 206), wherein universal supports based on nucleosidic and non-nucleosidic linkages have been employed in the synthetic protocol. However, a polymer support that would permit repetitive syntheses (of the order of 25 or more) leading to multiple oligonucleotides is still elusive.
Considering the wide potential of such supports, some efforts have been made in this direction. In one such report, Hardy et al. (Nucl. Acids Res., 1994, 22, 2998) have developed a method for producing more than one oligonucleotide per synthesis via a linker phosphoramidite called two oligomers per synthesis (TOPS). Using this reagent, two entirely different oligomers (synthesis of PCR primers) or multiple copies of a single oligomer could be generated in one continuous synthesis. After synthesis, aqueous ammonium hydroxide treatment releases either the pair of oligonucleotides or a single oligomer in large amount. The strategy further demands removal of terminal phosphate groups from the 3′-end, which requires harsh or prolonged deprotection conditions and non-quantitative dephosphorylation leaves unwanted impurities in the mixture.
In another report, Pon et al. (J. Org. Chem., 2002, 67, 856) have developed a simple strategy called tandem oligonucleotide synthesis. The approach is simplified in a way that it allows the synthesis of two different isolable oligonucleotides. In this approach, the first oligonucleotide is synthesized on a succinylated support and 5′-hydroxyl of the first oligonucleotide is used as the starting point for the synthesis of second oligonucleotide after introducing a base labile hydroquinone-O,O′-diacetyl (Q) linker at 5′-hydroxyl group. The oligonucleotides can be cleaved from the support in sequential manner, if required. However, this approach along with the above one are limited to producing two different oligomers or one oligomer multiple times on a single polymer support. Moreover, selective cleavage, in the latter one, often leads to the contamination of one oligonucleotide into another one, which prevents the use of the synthesized primers in DNA sequencing and requires purification prior to their use. Furthermore, carboxylate derivatives of all the four nucleosides are required.
In more recent communications, Pon et al. (Pon et al., Nucl. Acids Res., 1999, 27, 1541; J. Chem. Soc., Perkin Trans 1 2001, 2638) have reported (using LCAA-CPG support with generated hydroxyl groups on the surface and with the leader nucleoside molecule attached through a labile Q-linker) carrying out six cycles of oligonucleotide synthesis, albeit support functionalization with appropriately protected nucleosides has been claimed for 25 cycles. However, this system inherently admits of a limitation insofar as CPG is vulnerable to alkaline conditions and that repeated exposures to alkaline conditions (aq. ammonium hydroxide/methylamine) could affect the morphology of the glass support. They noticed this observation while using the reusable support for oligonucleotide synthesis in the seventh cycle and reported it in their later publication (J. Chem. Soc., Perkin Trans 1, 2000, 2638). Moreover, the above methodology also makes use of a non-conventional capping reagent (chloroacetic anhydride/methoxyacetic anhydride/p-tert-butylphenoxyacetic anhydride) for blocking residual hydroxyl groups on the polymer support. Moreover, at least four different pre-derivatized supports (dA, dC, dG and dT) would be required for all kinds of oligodeoxyribonucleotide synthesis. More recently, in yet another attempt, Pon and Yu have demonstrated the use of a new class of linker phosphoramidites (containing a cleavable 3′-ester linkage), for functionalization of supports in the machine itself. However, in this case also, the preparation of four nucleoside-linker phosphoramidites and the requirement of four additional ports in the machine for coupling of these linker molecules are the main limitations.
Therefore, there is a need to design a new polymer support, where (i) the preparation of all the four nucleoside (dA, dC, dG and T) derivatized polymer supports could be avoided, (ii) the oligomer sequences could be cleaved under non-ammoniacal conditions from the support, (iii) the cleaved support could easily be refunctionalized for repetitive oligonucleotide syntheses, (iv) the functionalization as well as oligomer cleavage procedures should be simple and straight-forward, and (v) fully deprotected oligomers could be obtained in single step. By taking these parameters in to consideration, the inventors have designed and synthesized a polymer support bearing a non-nucleosidic universal linker and a non-ammoniacal cleavable linker to avoid surface erosion during cleavage of oligomer sequences from the support.